EFFECTS OF CHLOROGENIC ACID (CGA) ON mRNA EXPRESSION OF INTERLEUKIN-6 (IL-6), TOLL-LIKE RECEPTOR 4 (TLR-4), AND MACROPHAGE MARKER EXPRESSION IN THE LIVER OF STREPTOZOTOCIN-INDUCED DIABETES RAT MODEL

GRECIA DEVINA S, dr. Ratih Yuniartha, Ph.D.; Wiwit Ananda Wahyu Setyaningsih, S.Keb., M.Sc.

2022 | Skripsi | S1 KEDOKTERAN

Latar Belakang: DM merupakan penyakit metabolik kronis yang berhubungan dengan hiperglikemia. Kondisi ini memicu stres oksidatif yang menyebabkan inflamasi, yang menyebabkan kerusakan organ, termasuk hati. Inflamasi hati dimediasi oleh molekul pro-inflamasi termasuk TLR-4, IL-6, dan makrofag. Chlorogenic Acid (CGA) dikenal karena kapasitas antioksidan dan anti-inflamasinya. Namun, masih sedikit informasi mengenai peran CGA terhadap inflamasi hati pada DM. Tujuan: Untuk mengetahui peran CGA terhadap inflamasi hati pada tikus Wistar diabetes, dengan mengamati ekspresi mRNA TLR-4, IL-6, dan makrofag (anti-CD68). Metode: Tikus Wistar jantan (n= 30, umur= 3 bulan) dialokasikan ke dalam kelompok kontrol, kelompok DM dimana STZ 60 mg/kgBB diinjeksi secara intraperitoneal kemudian diamati selama 6 minggu (DM1.5, n=5) dan 8 minggu (DM2, n=5). Kelompok DM1.5 diberikan tiga dosis CGA yang berbeda, yaitu 12,5 mg/kgBB (CGA1, n=5), 25 mg/kgBB (CGA2, n=4), dan 50 mg/kgBB (CGA3, n=6), sekali sehari selama empat belas hari. Pada akhir penelitian, tikus diterminasi dan spesimen hati diambil untuk ekstraksi RNA. Ekspresi mRNA TLR-4 dan IL-6 diukur dengan Reverse Transcription-Polymerase Chain Reaction (RT-PCR), sedangkan pewarnaan imunohistokimia dengan anti-CD68 digunakan untuk mendeteksi keberadaan makrofag. Hasil: Kelompok DM2 menunjukkan kadar glukosa, ekspresi mRNA TLR-4 dan IL-6 yang lebih tinggi secara signifikan dibandingkan kelompok kontrol. Terdapat infiltrasi makrofag yang lebih banyak pada kelompok DM1.5 dan DM2. Kelompok perlakuan CGA, terutama CGA1, secara signifikan menunjukkan kadar glukosa darah dan ekspresi mRNA TLR-4 yang lebih rendah dibandingkan dengan kelompok DM2. Terlihat penurunan infiltrasi makrofag pada kelompok CGA1 dan CGA2 dibandingkan dengan kelompok DM. Sementara itu, tidak ada perbedaan signifikan yang diamati pada ekspresi mRNA IL-6 pada kelompok perlakuan CGA dibandingkan dengan kelompok DM. Kesimpulan: CGA dengan dosis 12,5 mg/kgBB berpotensi untuk meredakan inflamasi hati pada kondisi diabetes melalui penurunan regulasi ekspresi mRNA TLR-4 dan makrofag.

Background: DM is a chronic metabolic disease associated with hyperglycaemia. This condition incites oxidative stress which causes inflammation, leading to organ damage, including the liver. Liver inflammation is mediated by pro-inflammatory molecules including TLR-4, IL-6, and macrophage. Chlorogenic Acid (CGA) is known for its antioxidant and anti-inflammatory capacities. However, there is little information regarding the role of CGA towards liver inflammation in DM. Aim: To investigate the role of CGA towards liver inflammation in diabetic Wistar rats, by observing the mRNA expression of TLR-4, IL-6, and macrophage (anti-CD68). Method: Male Wistar rats (n= 30, age= 3 months old) are allocated into control group, DM groups where 60 mg/Kg body weight (BW) of STZ were intraperitoneally injected and then observed for 6 weeks (DM1.5, n=5) and 8 weeks (DM2, n=5). DM1.5 group was given three different doses of CGA, which were 12.5 mg/kgBW (CGA1, n=5), 25 mg/kgBW (CGA2, n=4), and 50 mg/kgBW (CGA3, n=6), once daily for fourteen days. In the end of the study, the rats were terminated and liver specimens were harvested for RNA extraction. The mRNA expression of TLR-4 and IL-6 were measured by RT-PCR, while immunohistochemistry staining with anti-CD68 was used to detect the presence of macrophage. Result: DM2 group showed significantly higher blood glucose levels, TLR-4 and IL-6 mRNA expression compared to control group. The liver of DM1.5 and DM2 group showed more abundant macrophage infiltration. CGA treated groups, especially CGA1, significantly lower blood glucose level and TLR-4 mRNA expression compared to DM2 group. Lower macrophage infiltration was observed in CGA1 and CGA2 group compared to DM group. Meanwhile, no significant differences were observed in IL-6 mRNA expression in CGA group compared to DM group. Conclusion: CGA with dosage 12.5 mg/kgBW have the potential to attenuate liver inflammation in DM through downregulation of TLR-4 mRNA expression and macrophage.

Kata Kunci : Chlorogenic Acid, Diabetes Mellitus, Liver Inflammation, Toll-Like Receptor 4, Interleukin 6, Macrophages.